*3.2.4. Prediction of amino acid residues involved in virus binding and host–virus specificity*

192 New Approaches to the Study of Marine Mammals

three marine mammals.

bonds are shown in yellow.

the virus, because the side-chains of these residues are closely located to those of viral H protein in a crystal structure of the complex [56]. Particularly, the residues at 127-131 are thought to form an intramolecular β-sheet with the β-strand of MV H [56]. The overall 3D structures of the interfaces are similar among SLAMs, but several among the total 32 amino acid residues possibly contributing the binding affinity to the virus, differed among the

The interfaces are viewed from the direction shown by the thick red arrow in Figure 3. The amino acid residues that possibly interact with the viral H protein, are shown with their position numbers. The different amino acid residues among the three mammal SLAMs are indicated in black, and their side-chains are shown with the atoms colored (black for carbons, blue for nitrogens, and red for oxygens). The shared residues among the three are shown in blue. Disulfide

**Figure 4.** Ribbon diagram of the 3D structure models of the SLAM interface for binding morbillivirus

from the spotted seal (a), Pacific white-sided dolphin (b), and manatee (c).

To identify amino acid residues that are important for host–virus specificity, we compared the 32 residues with those of land mammal SLAMs (Table 2). The difference in the SLAM interface was only two amino acid residues between seal and dog (Val and Ile at position 74, and Arg and Gln at position 129), between human and marmoset (Leu and Phe at position 119, Val and Ile at position 126, marmoset data not shown), and between cow and sheep (Asp and Gly at position 87, and Arg and His at position 90). This is consistent with the evidence that mass die-offs of Baikal seals and Caspian seals were caused by CDV; marmosets are highly sensitive to MV; and that RPV and PPRV can infect ruminants. It is noted that the identity of these 32 residues between dolphin and cow SLAMs is very high, although they are infected by different morbilliviruses, CMV and RPV, respectively. Four residues are different between the two animals, while eleven and fourteen residues are different between dolphin and seal, and between dolphin and humans, respectively.



Host-Virus Specificity of the Morbillivirus Receptor, SLAM, in Marine Mammals:

residues are located spatially near each other in the 3D homology model of the interface (Figure 4). To clarify the influence of the changes in the charge, electrostatic potentials on the surface of SLAM interfaces of the three marine mammals, are shown in Figure 5. It can be seen that the zone constituted by the residues at positions 84, 85, 87, and 90 are different

Electrostatic potential was calculated using DelPhi v.4 [77,78]. Positively and negatively charged surfaces are colored

**Figure 5.** Electrostatic potential on the surface of the SLAM interfaces with morbilliviruses deduced from the 3D homology model structures of the SLAM interfaces from the spotted seal (a), Pacific white-

The amino acid residues at positions 67 and 68 are also highly variable among the three interfaces (Table 2). The manatee-specific residue E67 appears to contribute greatly to the formation of the negatively charged zone (Figure 5). The manatee SLAM has two another specific residues, R61 and P90, which induce an acquisition of stronger positive charge or a loss of positive charge on the interface (Table 2, Figure 4). These findings suggest that if there is a morbillivirus for the sirenians, it has the H protein with a very different SLAM

Crystallographic analysis of the complex of MV H and marmoset SLAM V unexpectedly showed two different potentially tetramic configurations, form I and form II [56]. Residue N53 is located at the interface only in form II. Its replacement mutant changing to Q53 showed a reduction in molecular masses, meaning the loss of glycosylation, and an unexpected increase in MV entry into human cells. The reason for this is not fully understood. However, because only primate and manatee SLAMs possess residue N53, it

among the SLAM interfaces of three marine mammals.

blue and red, respectively.

binding interface.

sided dolphin (b), and manatee (c).

may also be involved in host–virus specificity.

Risk Assessment of Infection Based on Three-Dimensional Models 195

The residue position, which varies among animals, is shaded. The light- and dark-shaded boxes indicate a variation in chemically (charge, hydrophilicity, etc.) similar or different residues. The asterisk indicates the specific residue with a chemical change for the animal SLAM. See the legend of Figure 2 for the animal names and accession numbers of the amino acid sequences used in this table.

**Table 2.** Amino acid residues on the SLAM interface possibly involved in regulating the binding and specificity of morbilliviruses.

Among the 32 residue positions, variations in amino acids were found at 18 positions (Table 2, light and dark shading). At six positions (63,73,74,119,121, and 126), the changes are between chemically similar residues (light-shaded boxes in Table 2) and these do not seem to markedly affect binding with the viruses. On the other hand, the variations at the other twelve residue positions (61,67,68,72,75,76,84,85,87,90,129, and 130; dark-shaded boxes in Table 2) occur in amino acids with chemically different characteristics. In particular, the variations among amino acids with opposite charge may significantly alter the affinity for viruses (positions 68,75, and 85). The twelve amino acid residues are thought to be important in determining host–virus specificity. Almost of the twelve residues are located in the edge region of the interface. This may indicate that residues located in the central region of the interface play an important role in virus entry itself, rather than in host–virus specificity. Alternatively, they may be essential for a primary immunological function.

A detailed binding assay using surface plasmon resonance analysis was carried out between human SLAM mutants and the MV H protein [56,57]. The respective changes in the residues from H61, E123, and R130 of the human SLAM interface to serine residues completely abolished the binding ability to the MV H protein. In crystallographic analysis, R130 was suggested to form an intramolecular salt bridge with E75 [56]. Only human SLAM possesses these two residues on the interface. As shown in Table 2, K68, a strong positively charged residue, is also specific for human SLAM. These facts suggest that they are key residues for MV infection. On the other hand, H61 and E123 are conserved in all mammals, except for R61 of manatees, suggesting that these residues play a crucial role in viral infection, rather than in host–virus specificity.

Although a detailed analysis of SLAM–virus interaction has not been conducted in systems other than the human SLAM–MV complex, animal-specific residues can be seen in Table 2. For example, the markedly specific residue set R84-K85–D87-H90 is found in dolphin. These residues are located spatially near each other in the 3D homology model of the interface (Figure 4). To clarify the influence of the changes in the charge, electrostatic potentials on the surface of SLAM interfaces of the three marine mammals, are shown in Figure 5. It can be seen that the zone constituted by the residues at positions 84, 85, 87, and 90 are different among the SLAM interfaces of three marine mammals.

194 New Approaches to the Study of Marine Mammals

Viruses PDV,

CDV

amino acid sequences used in this table.

specificity of morbilliviruses.

than in host–virus specificity.

a.a. No. Seal Dog Dolphin Cow Sheep Human Manatee 125 N N N N N N N 126 F F I V V V F 127 S S S S S S S 128 V V V V V V V 129 R\* Q Q Q Q Q Q 130 H H Q H H R Q 131 F F F F F F F

PPRV

The residue position, which varies among animals, is shaded. The light- and dark-shaded boxes indicate a variation in chemically (charge, hydrophilicity, etc.) similar or different residues. The asterisk indicates the specific residue with a chemical change for the animal SLAM. See the legend of Figure 2 for the animal names and accession numbers of the

**Table 2.** Amino acid residues on the SLAM interface possibly involved in regulating the binding and

Among the 32 residue positions, variations in amino acids were found at 18 positions (Table 2, light and dark shading). At six positions (63,73,74,119,121, and 126), the changes are between chemically similar residues (light-shaded boxes in Table 2) and these do not seem to markedly affect binding with the viruses. On the other hand, the variations at the other twelve residue positions (61,67,68,72,75,76,84,85,87,90,129, and 130; dark-shaded boxes in Table 2) occur in amino acids with chemically different characteristics. In particular, the variations among amino acids with opposite charge may significantly alter the affinity for viruses (positions 68,75, and 85). The twelve amino acid residues are thought to be important in determining host–virus specificity. Almost of the twelve residues are located in the edge region of the interface. This may indicate that residues located in the central region of the interface play an important role in virus entry itself, rather than in host–virus specificity. Alternatively, they may be essential for a primary immunological function.

A detailed binding assay using surface plasmon resonance analysis was carried out between human SLAM mutants and the MV H protein [56,57]. The respective changes in the residues from H61, E123, and R130 of the human SLAM interface to serine residues completely abolished the binding ability to the MV H protein. In crystallographic analysis, R130 was suggested to form an intramolecular salt bridge with E75 [56]. Only human SLAM possesses these two residues on the interface. As shown in Table 2, K68, a strong positively charged residue, is also specific for human SLAM. These facts suggest that they are key residues for MV infection. On the other hand, H61 and E123 are conserved in all mammals, except for R61 of manatees, suggesting that these residues play a crucial role in viral infection, rather

Although a detailed analysis of SLAM–virus interaction has not been conducted in systems other than the human SLAM–MV complex, animal-specific residues can be seen in Table 2. For example, the markedly specific residue set R84-K85–D87-H90 is found in dolphin. These

PPRV, RPV

MV None

CDV CMV RPV,

Electrostatic potential was calculated using DelPhi v.4 [77,78]. Positively and negatively charged surfaces are colored blue and red, respectively.

**Figure 5.** Electrostatic potential on the surface of the SLAM interfaces with morbilliviruses deduced from the 3D homology model structures of the SLAM interfaces from the spotted seal (a), Pacific whitesided dolphin (b), and manatee (c).

The amino acid residues at positions 67 and 68 are also highly variable among the three interfaces (Table 2). The manatee-specific residue E67 appears to contribute greatly to the formation of the negatively charged zone (Figure 5). The manatee SLAM has two another specific residues, R61 and P90, which induce an acquisition of stronger positive charge or a loss of positive charge on the interface (Table 2, Figure 4). These findings suggest that if there is a morbillivirus for the sirenians, it has the H protein with a very different SLAM binding interface.

Crystallographic analysis of the complex of MV H and marmoset SLAM V unexpectedly showed two different potentially tetramic configurations, form I and form II [56]. Residue N53 is located at the interface only in form II. Its replacement mutant changing to Q53 showed a reduction in molecular masses, meaning the loss of glycosylation, and an unexpected increase in MV entry into human cells. The reason for this is not fully understood. However, because only primate and manatee SLAMs possess residue N53, it may also be involved in host–virus specificity.
